bacterial contamination in selected commercially important

Bacterial Contamination in Selected Commercially Important Bivalve Species

and Farmed Seaweed in the Panguil Bay, Northern Mindanao

Sonia M. Dejarme, Emilie G. Tubio and Mariefe B. Quiñones

Mindanao State University at Naawan, 9023 Naawan, Misamis Oriental

Email: [email protected]

ABSTRACT

Bivalve molluscs comprise an important artisanal invertebrate fishery in Panguil Bay in

northern Mindanao. The bay also supports one of the largest seaweed farming projects in the

region. Recent concerns on declining water quality in the bay due to domestic sewage and

industrial effluents motivated this research into investigating the bacterial contamination of food

resources from the bay. Ten selected commercially important edible bivalve species from wild

population and Kappaphycus alvarezii (guso) from a seaweed farm were collected monthly for

12 months from the Lanao del Norte side of Panguil Bay for the analysis of bacterial

contamination, particularly fecal coliform bacteria. Water and sediments from the habitats of these organisms were also sampled for the same microbiological analyses. Fecal coliform

bacteria were noted in Arca antiquata (litob-litob), Katylesia sp. (punaw), Meretrix meretrix

(burnay), and Modiolus metcalfei (amahong) from freshly hand-picked samples from the muddy tidal flat in certain areas in the municipalities of Lala and Baroy, Lanao del Norte. Bivalve

samples from market displays such as Anadara sp. (balinsala), Modiolus sp. (baluyang),

Pharella acutidens (tudlo-datu), and Trisidos sp. (lubag-lubag), Anodontia edentula (imbaw) and Mercenaria sp. (tuway) were also similarly contaminated. The extent of bacterial contamination

from hand-picked and market samples exceeded the current acceptability standards of Philippine

bivalves for human consumption as set by the Bureau of Food and Drugs (BFAD). It is strongly

recommended that bivalves from Panguil Bay be depurated and cooked properly before these are served in dining tables. There is no standard for fecal coliform density in seaweeds but the

values obtained in the farmed seaweed, K. alvarezii, was within the acceptability standard for

Philippine bivalves, thus, it may be considered safe for human consumption.

Keywords: Panguil Bay, bacterial contamination, fecal coliform, edible bivalves, farmed

Kappaphycus

INTRODUCTION

Panguil Bay in Northwestern Mindanao is known for its rich fishery resources of prawns,

crabs and molluscs. Bivalves are the most abundant shellfish or mollusc in shallow coastal

waters near the crowded fishing villages of Lala, Baroy, Tubod and Kolambugan in the Lanao

del Norte side of the Bay. The seaweed Kappaphycus, which does not grow naturally in Panguil

Bay, has been successfully cultured at a commercial scale in the coastal areas of Kolambugan

and Tubod.

mailto:[email protected]

Dejarme, et al.: Bacterial contamination… 43

Bivalve shellfish and farmed seaweed are economically valuable sources of proteins, vitamins and minerals for coastal communities in Panguil Bay, however, the lack of proper

disposal facilities by many households contributes to declining water quality in the bay.

Domestic sewage, feces of human and other warm-blooded animals, normally contains

pathogenic bacteria, viruses, protozoans and fungi. When discharged into coastal waters, it can cause microbial contamination of filter-feeding organisms such as bivalves. Seaweeds can also

become contaminated when colloidal particles from domestic sewage become jammed or

embedded into the narrow forks of branching seaweed thalli. Eating these contaminated food products raw or only partially cooked presents a serious risk to human health.

Biological pollution occurs when microorganisms enter a water body from sources such

as human waste, food operations, meat packing plants and medical facilities. Coliform, fecal

coliform, and non-coliform bacteria such as fecal streptococcus are the major types of bacteria in polluted waters and are generally harmless to man (Tchobanoglous, 1979). Certain pathogens

most commonly found in animal feces, however, are causal agents of human diseases such as

food poisoning, diarrhea, typhoid fever, hepatitis A and many more (West, 1989). Bivalves such

as oysters, mussels and clams are the most significant groups of shellfish associated with gastro- enteritis because of their filter-feeding activity (West, 1985). Contamination of shellfishes by

pathogenic microorganisms is not, however, confined to sewage disposal since pathogens such as

Vibrio and Aeromonas thrive in natural aquatic environments (Morris and Black, 1985). Contamination of shellfish viscera by pathogenic microorganisms can present a significant health

risk to people who consume them raw or lightly cooked.

The presence of fecal coliform bacteria is commonly used as an indicator to assess

potential health hazards to consumers of raw shellfish, the effectiveness of depuration and cooking, and the effects of sewage disposal on the aquatic environment. Fecal coliform also

indicates potential contamination by pathogenic microorganisms not only from humans but also

from other warm-blooded animals. Reports on bacterial contamination of shellfishes in the Philippines, however, are few and have been confined to few species such as oysters and mussels

(Gacutan, et al. 1986). Some works have been published on bacterial depuration to reduce high

bacterial and virus levels to acceptable ones (Dore and Lees, 1995; Lee, et al. 2008). Likewise

very little work on bacterial contamination of seaweed is available and mostly focused on “ice- ice” disease (Largo et al. 1995; Tisera and Naguit, 2009). This study was conducted to determine

the extent of bacterial contamination, particularly of fecal coliform, on bivalves and farmed red

seaweed harvested from Panguil Bay, and to recommend strategies to reduce its potential impact on public health.

MATERIALS AND METHODS

Collection site for bivalve samples

Panguil Bay is located in the northwestern part of Mindanao (between 7°51' to 8°12' N;

123°37' to 123°55' E) and is a shared resource of the provinces of Lanao del Norte, Zamboanga

del Sur, and Misamis Occidental (Fig.1). Bivalve samples were collected from the littoral zones

of four sites, namely, Pacita, Lala; Raw-an Pt., Baroy; Poblacion,Tubod; and Mukas,

44 J. Environmental Science and Environment, Vol. 3 (2015)

Kolambugan (black circles in Fig. 1). Pacita, Lala and Raw-an Pt., Baroy (Fig. 2) are major

gathering grounds of both traditional and commercial bivalve gatherers and are located close to fishing villages with adequate toilet facilities. Bivalve samples from these sites were handpicked

by the project team while bivalves from Tubod and Mukas were obtained from market displays.

Figure 1. Map of Panguil Bay showing the sampling sites for bivalves (black circles) and

farmed seaweed (green square).

Collection site for farmed Seaweeds

The source of farmed seaweed samples of K. alvarezii (locally called guso) used in this

study is the commercial seaweed farm in the coastal waters of Simbuco, Kolambugan, Lanao del

Norte (open circle in Fig. 1) The farm site covers about 300 hectares and is located about 100 m from shore at a depth of 3-6 m (Fig. 2). The shoreline of Simbuco near the guso farm site is

densely populated where people mostly lived in stilt houses with inadequate toilets.

Sample collection

At least two liters of samples of selected commercially important bivalves (from shellfish

gatherers and market vendors) and two kilograms of farmed seaweed thalli were obtained

monthly from May 2003 to April 2004 and transported to MSU-Naawan following standard protocols in handling and transport of samples (Al-Jebouri, 1981; West, 1988; APHA, 1998).

One-kg samples of water and sediment in each site were also collected.


Figure 2. Collection sites for bivalve (top panel) and seaweed (bottom panel) samples in Lanao

del Norte.

Laboratory analysis

Sample processing. Random samples of 30 -100 pieces from each pack of bivalve

samples were rinsed with sterile seawater and processed for analysis immediately upon arrival in

the laboratory following the procedure described by Al-Jebouri (1981) and Hasegawa (1987). The bivalves were pried open and shucked using a sterilized knife. Exactly 50 g of bivalve meat

were aseptically weighed in an electronic top loading balance and homogenized in a sterile

blender for 2-5 minutes. The homogenate was mixed with 450 ml sterilized seawater to produce

500 ml of bivalve homogenate of 1:10 dilution. The homogenate was transferred into a 1000-ml capacity sterile beaker and was serially diluted and inoculated to appropriate medium (Fig. 3). A

random sample from the two-kilogram seaweed thalli was cut into small pieces then 50g of

cuttings were homogenized in precisely the same manner as the bivalve preparation. A 50-gram aliquot sample from each 1kg sediment and water samples was likewise prepared for analysis.

Total Bacterial Density (Heterotrophic Plate Count)

The bacterial densities (expressed as colony-forming units per mL or CFU

-mL) of the

different samples of bivalves, seaweeds, water and sediments were estimated using the pour plate

method following the standard APHA (1998) protocol for bacteriological analysis.


Figure 3. Flow chart of methods in sample preparation and enumeration of

total and fecal coliform.

Estimation of Coliform Load

The Multiple-Tube Fermentation Technique was used to estimate the total and fecal

coliform bacteria in the samples. This consisted of three tests, namely; Presumptive Test, Confirmed Test and the Completed Test following standard procedures (Refai, 1979; Hasegawa,

1987; Greenberg et al., 1992; APHA, 1998) (Fig. 3). The densities of total coliform and fecal

coliform bacteria were estimated based on tabulated MPN Indexes (0.1, 0.01 and 0.001) for 100 g food and other solid samples (Andrews, 1992) and 10.0, 1.0 and 0.01 for 100 mL water

samples (Greenberg et al., 1992; and APHA, 1998).

Depuration of Bivalves

Bacteria can be removed from bivalves in different ways, a procedure called depuration.

In Panguil Bay and other Philippine fishing villages a local practice called laming is a practical

and effective process in removing impurities from edible bivalves. The bivalves are placed in a pail or basin filled with filtered seawater or brackish water for 24-48 hrs. During this period the

water is drained and replenished four to six times because bacteria accumulated by the bivalve

are flushed out through the feces. A test was conducted to determine the efficiency of the said

practice in removing fecal coliform and other bacteria. One can (1-liter cap) sample of each bivalve species was placed in separate basins filled with filtered seawater and provided with

aeration at the wet laboratory of MSU-Naawan. The filtered seawater was changed six times for

48 hrs. Then the purified bivalves were processed for microbiological analyses to determine the bacterial load.


RESULTS AND DISCUSSION

Ten edible bivalve species from different habitat types and one red seaweed were

sampled in the Lanao side of Panguil Bay (Table 1).

Table 1. List of bivalve shellfish and seaweed used in the assessment of bacterial contamination

in Panguil Bay.

Local Name English Name Scientific Name Collection Site Habitat Characteristics

Litob-litob Blood clam Arca antiquata Lala, Pacita Muddy tidal flat Punaw Hen clam Katylesia sp. Lala, Pacita Muddy tidal flat

Burnay Surf clam Meretrix meretrix Lala, Pacita Muddy tidal flat Amahong Brown mussel Modiolus metcalfei Lala, Pacita Muddy tidal flat

Punaw Hen clam Katylesia sp. Raw-an, Baroy Sandy tidal flat

Burnay Surf clam Meretrix meretrix Raw-an, Baroy Muddy and sandy tidal flats

Balinsala Cockle Anadara sp. Tubod market Deep coastal waters of Tubod

Baluyang Big Brown Modiolus sp. Tubod market Deep coastal waters of Tubod

Mussel Tudlo-datu Razor or Jacknife Pharella acutidens Tubod market Muddy substrate of Tubod

clam Lubag-lubag Twisted Shell; Trisidos sp. Tubod market Deep coastal waters of Tubod

Propeller Ark Imbaw Mangrove clam Anodontia edentula Mukas market Muddy substrate of Kolambugan Tuway Mud clam Mercenaria sp. Mukas market Muddy substrate of Kolambugan

Guso Red Seaweed Kappaphycus Simbuco Deep coastal water farm site of

alvarezii Kolambugan

Bacterial Density

Bacterial densities (CFU-g), total coliform and fecal coliform (MPN

-100g or MPN

-100mL) in

bivalves and other samples varied with species, sampling months and sampling areas. Average

bacterial density in freshly hand-picked bivalves was highest in M. metcalfei (720 x 103CFU

-g)

from the muddy tidal flat of Pacita, Lala while Katylesia sp. from the sandy substratum of Raw-

an Pt., Baroy had the highest bacterial density (903 x 103CFU

-g) (Table 2). Bacterial load may

also vary according to body size, as in the case of M. meretrix where medium-sized specimens had higher bacterial count than smaller ones gathered from Lala. On the average across sites, bivalves from Raw-an Pt., Baroy had higher total bacterial accumulation where both water and sediment had higher bacterial load (Table 2).

An extremely high bacterial density was observed in Mercenaria sp. (tuway) collected

from Mukas market (>6647 x 103CFU

-g) while a lower density (548 x 10

3CFU

-g) was obtained

from a closely related mangrove clam, Anodontia edentula (Table 3). The bivalves obtained from Tubod market generally had high mean bacterial density with the highest value of 1,109 x

103CFU

-g obtained from P. acutidens. The brown mussel Modiolus sp., on the other hand, had a

very low bacterial density (3.02 x 103CFU

-g) although its location in the deeper, offshore areas

of Tubod may have reduced the level of contamination (Table 1).


Anodontia edentula 548 34

Mercenaria sp. >6647* 165

Table 2. Average bacterial density (CFU-g

or CFU-ml

) and fecal coliform geometric mean (xg) of

MPN-100g

or MPN-100ml

in freshly hand-picked bivalves, seaweed, water and sediment samples collected from May 2003-April 2004.

Sampling sites/Samples Bacterial Density

(x103 CFU

-g or CFU

-ml)

Fecal coliform

(xg)

Lala

Arca antiquate 593 77

Katylesia sp. 448 178

Meretrix meretrix 512 79

Modiolus metcalfei 720 33

Water 13.9 84

Sediment 242 5.83

Raw-an Pt.

Katylesia sp. 903 54

Meretrix meretrix (medium) 781 179

Meretrix meretrix (small) 401 24

Water 316 159

Sediment 845 23

Simbuco

Kappaphycus alvarezii 760 4

Water 68 43

Table 3. Average bacterial density (x103CFU

-g or CFU

-ml) and fecal coliform geometric

mean(xg) of MPN-100g

or MPN-100ml

in market collection of bivalve samples from May 2003- April 2004.

Sampling sites/Samples Bacterial Density

(x103

CFU-g

or CFU-ml

)

Fecal Coliform Density

(xg)

Tubod

Anadara sp. 930 2

Modiolus sp. 3.02 <2

Pharella acutidens 1109 85

Trisidos sp. 525 2

Mukas

* - Number of colonies exceed 300 colonies per plate.

It is not within the scope of the study to differentiate the bacterial sources or determine what percentage of the bacteria comes from the normal aquatic flora and those from domestic

sewage contamination. The lower bacterial density in water than that of the sediment could probably be due to the high organic matter accumulated in the sediment. The data also suggest,

rather inconclusively, that muddy substrates tend to accumulate higher bacterial density than

sandy substrates.

Bivalves are predominantly filter feeders. This feeding mechanism results in the natural

ability to extract bacteria from the coastal waters and may be variable across species. Among the


hand-picked bivalves Katylesia sp., M. meretrix, and M. metcalfei had the highest bacterial load. This could not be attributed to the habitat characteristics alone because M. meretrix, and M.

metcalfei are muddy substrate dwellers while Katylesia sp. can be found both in sandy and

muddy sediments. It would be an interesting future study to determine the filtration capacity of

different bivalve species and their accumulation of bacteria in the internal organs.

The bacterial load of bivalves from the local markets varied widely with opposite

extreme values, with the lowest obtained from Modiolus sp. and the highest from Mercenaria

sp. Differences in habitat characteristics of these two organisms could have greatly influenced the wide discrepancy in their bacterial load. Modiolus sp. inhabits the deeper portion of Panguil

Bay, relatively far from the influence of domestic waste. Mercenaria sp., on the other hand,

abounds in deep, muddy mangrove areas in the densely populated and sanitation-poor area of

Mukas. The prop roots and pneumatophores of different mangrove species are quite effective in trapping and accumulating large volume of domestic and agricultural wastes from lowland and

upland areas.

Despite the low bacterial population in Simbuco waters (68 x 103

CFU-g

), thalli of farmed

K. alvarezii were found to have a high bacterial density (760 x 103

CFU-g

). This result suggests

the ability of the seaweed to accumulate bacteria from water. In the seaweed farms of Simbuco, thalli of K. alvarezii are closely tied along the length of a rope (80-100 m each) hanging between two posts. The seaweed farms are adjacent each other so floating organic debris and fresh solid waste from domestic sources can become easily entangled in the mesh of thalli-laden

ropes.

Fecal Coliform Density

All bivalve samples collected from different sources for this study were contaminated with fecal coliform (Table 2 and 3) and even some with Escherichia coli. The most contaminated bivalves from all source are M. meretrix (from Raw-an Pt.), Katylesia sp. (Pacita, Lala) and

Mercenaria sp. (Mukas market) at 179 MPN-100g

, 178 MPN-100g

and 165 MPN-100g

, respectively (Fig. 4). The lowest concentration of fecal coliform was observed in M. metcalfei (Lala) and M. meretrix (Raw-an Pt). The farmed seaweed K. alvarezii had a very low fecal coliform content (4

MPN-100g

), .

The coliform bacterial composition varied with the sample group (bivalve and seaweed),

sampling area and sampling month as indicated in the IMViC Test (Indole, Methyl red, Voges-

Proskauer and Citrate) for preliminary differentiation of coliform and fecal coliform groups. Two

coliform species (Aeromonas sp. and Pseudomonas sp.) and seven fecal coliform species (Citrobacter freundii, Citrobacter sp., Enterobacter aerogenes, Escherichia coli, Escherichia

hermanii, Escherichia vulneris and Klebsiella sp.) were identified from the samples. Gram

staining also showed that bacterial morphology varied from short rod to rod-shaped, non-spore- forming bacteria.


A. Meretrix meretrix B. Katylesia sp.

C. Mercenaria sp . D. Pharella acutidens

Figure 4. Freshly handpicked (A&B) and market display (C&D) bivalves in Panguil Bay

with the highest fecal coliform content.

High fecal coliform densities were generally observed in October, November, December,

January, February and April. These were rainy months (except in late April) and unusual

volumes of water runoff were noted in the sampling sites which must have washed away human feces and that of other warm-blooded animals and brought these to the sea. The close proximity

of the bivalve beds to residential areas makes them highly vulnerable to fecal coliform. Davis et

al. (1980) declared that the aquatic environment is physiologically hostile to fecal bacteria, thus, these microorganisms are not expected to grow normally in the salty coastal waters. High fecal

coliform density in many bivalve species could be due to recent or continuing fecal

contamination rather than bacterial reproduction or regrowth (Zaen–Ul-Abedin, undated). In

contrast, water is the usually the first recipient of domestic sewage from terrestrial sources which would explain higher fecal coliform density in the water around Raw-an Pt. than some bivalves.

Depuration or Self-Purification in Bivalves

The common practice of depuration or laming of freshly gathered bivalves serve to

reduce bacterial load through regular excretions or ‘self-purification’. The experimental depuration of different bivalves for only two days reduced bacterial density between 17.64%

(M. meretrix from Lala) to as much as 98.64% (M. metcalfei from Lala). Depuration also

reduced total coliform and fecal coliform densities in this bivalve species by 81.25%-99.51% and


Katylesia sp. (Lala) 567 1660 -192.77 ≥1600 500 68.75 ≥1600 300 81.25

Mercenaria sp. (Mukas) 2890 3000 -3.81 ≥1600 300 81.25 500 300 40.00

Pharella acutidens (Tubod) 1640 2360 -43.90 ≥1600 600 62.50 1600 500 68.75

40.0%-99.51%. Mortalities occurred in some species during depuration which increased bacterial activity, but overall depuration or the local ‘laming’ practice can remove most of the bacterial

load and fecal coliform in commercially gathered bivalves, and thus, safeguard health of the

consuming public. Reduction in fecal coliform was more efficient than the removal of total

bacterial load, a finding that supports expert opinion that the presence of fecal coliform in bivalves is mainly due to recontamination.

Table 4. Comparison and percentage reduction of bacterial densities (x 10

3 CFU

-g); total

coliform (MPN-100g

) and fecal coliform (MPN-100g

in non-depurated (ND) and depurated (D) bivalves.

Samples Bacterial Densities

ND D %

ND

Total Coliform

D %

Fecal Coliform

ND D %

M. meretrix (Lala) 567 467 17.64 1600 7.8 99.51 1600 7.8 99.51 M. meretrix (Raw-an, 430 35.2 91.81 ≥1600 32 98.00 300 32 89.33

Muddy) A. antiquata (Lala) 154 71.0 53.90 ≥1600 210 86.88 300 7.0 97.67

M. metcalfei (Lala) 236 3.21 98.64 1600 300 81.25 300 2.0 99.33

Katylesia sp. (Raw-an) 753 853 -13.28 ≥1600 64 96.00 1600 54 96.63

M. meretrix (Raw-an, 338 * ≥1600 17 98.94 110 7.8 92.91

Sandy)

Legend: (-) Increased bacterial density on depurated due to mussel mortality

* No analysis, >50% died during depuration process

Shellfish Standards for the Philippines

The current standards (Table 5) for bacterial load of Philippine shellfish, are 500,000

CFU-g

for fresh oysters and 100,000 CFU-g

for mussel and clams, and 20 MPN/100g for coliform load (BFAD). In the present study the densities of bacteria, coliform bacteria and fecal coliform in all the edible bivalves generally exceeded the current standards. Results generally show that coliform densities for seaweeds are low, however, a standard for bacterial and fecal coliform load for edible seaweeds is not available. If the current standards for shellfish will be adopted for seaweeds, then it is logical to say that it is safe to eat raw seaweed from Simbuco, but such an advice cannot encompass the farmed seaweed in Ozamiz City.

MANAGEMENT IMPLICATIONS AND RECOMMENDATIONS

This study provides strong evidence that the commercially important bivalves and

seaweed from Panguil Bay are generally contaminated with coliform and non-coliform bacteria, in fact, exceeding the Philippine standards for safe consumption of shellfish. Bacterial

contamination of seafood is chronic and pervasive in many harvesting areas and endangers

public health by passing the contamination to the consumers. Microbial contamination of

bivalves and seaweed is largely underestimated and undermanaged, and this oversight by the local government and the health department poses a potential public health risk. The elimination

of microbial contamination from consumer seaweed and bivalves in Panguil Bay and other bays

should be at the top priority agenda by government and civil society.


Table 5. Comparison of bacterial load and fecal coliform) in bivalves and BFAD (Bureau of

Food and Drug) Standards.

Bacterial

Bivalve species load

BFAD Std.

Bacterial load

Fecal

coliform

BFAD Std.

for coliform (x10

3 CFU

-g) (x10

3 CFU

-g) (MPN

-100g) (MPN

-100g)

Mussel & Clams

100

20

A. antiquata 593 77

Anadara sp. 930 2 A. edentula 548 34 Katylesia sp. (Lala) 448 178 Katylesia sp. (Raw-an) 903 54 M. meretrix (Lala) 512 79 M. meretrix (Raw-an muddy) 781 179 M. meretrix (Raw-an sandy) 401 24 M. metcalfei 720 33 Mercenaria sp. >6647* 165 Modiolus sp. 3.02 <2 Pharella acutidens 1109 85 Trisidos sp. 525 2

* Number of colonies exceeded 300 colonies per plate.

On the basis of these findings, it is recommended that consumers should first depurate

these bivalves and properly cook them to avoid ingesting pathogenic microorganisms. Despite the low levels of bacterial contamination in farmed seaweeds it is still important to wash the

thalii thoroughly with clean water to remove colloids, epiphytes and bacterial contaminants

before eating them raw as salad. Further research is also needed for the improvement of public

health control and the enhancement of shellfish safety.

ACKNOWLEDGEMENT

This study was realized through the support of MSU-Naawan which we gratefully

acknowledge here. We also thank the seaweed growers in Mukas, Lanao del Norte for sharing specimens of cultured Kappaphyccus and Daniel D. Gonzales for his assistance in the field and

laboratory activities.

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bacterial contamination in selected commercially important

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